This study was conducted to assess the impacts of prolonged fasting (70 and 120 days) on the morphological, biochemical, oxidative stress responses, immune-related gene expression, histopathology, and DNA damage in rainbow trout. Final weight (FW), hepatosomatic index (HSI), and condition factor (CF) significantly decreased in both 70 and 120 days of fasting compared to the pre-fasting group (p < 0.05). Fasting led to a significant reduction in serum blood metabolites (glucose, total protein, triglyceride, T. cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL)) and endogenous reserves (protein and lipid). However, plasma acetylcholinesterase (AChE) activity, aspartate aminotransferase (AST), alanine aminotransferase (ALT), interleukin (IL1), tumor necrosis factor (TNF1α), and transferrin (TF) increased significantly (p < 0.05). While malondialdehyde (MDA) levels compared to the pre-fasting group increased in the liver and muscle tissues (70 and 120 days), glutathione (GSH) enzyme activities decreased significantly in both tissues (p < 0.05). Histopathologically, both fasting groups (70 and 120 days) when compared to the pre-fasting group led to steatosis, necrosis and degeneration in hepatocytes, inflammation and hyperemia in the liver tissue and hyaline degeneration, atrophy, and inflammation in muscle tissue. Additionally, 8-OHdG levels of the liver and muscle tissues at 120 days’ fasting were more severe according to 70 days’ fasting. Finally, blood, the liver, and muscle tissues may be helpful to assess the impacts of fasting and fasting stress in rainbow trout.